The present invention relates to novel, non-peptidic small organic compounds having an affinity for cyclophilin (CyP)-type immunophilin proteins. The invention further relates to the uses of these compounds for binding CyP-type proteins, inhibiting their peptidyl-prolyl isomerase activity, and for research, development, and therapeutic applications in a variety of medical conditions.
Immunophilins are a group of proteins which are functionally characterized by their ability to bind certain immunosuppressive drugs. Two structurally and pharmacologically distinct classes of immunophilins are the FK506 binding proteins (FKBPs) and the cyclophilin (CyP) proteins. Although the prototypical members of these two protein families, FKBP12 and cyclophilin A, are both involved in the cellular mechanisms which mediate immunosuppression, they display selective affinities for very different types of immunosuppressants: members of the FKBP family bind to the macrolide antibiotics FK506 and rapamycin, whereas members of the CyP family bind to the cyclic undecapeptide Cyclosporin A (CsA).
Common to all immunosuppressant drugs is their ability to interfere with the intracellular signalling cascades of cells of the immune system. In the case of FK506 and CsA, binding of these drugs to their respective receptor proteins FKBP12 and cyclophilin A results in the cross-linking of the intracellular phosphatase calcineurin to the drug-receptor complex. The resulting inactivation of calcineurin eventually leads to the accumulation of phosphorylated calcineurin substrates, including the signaling protein NFAT (nuclear factor of activated T-cells). NFAT plays an important role in the regulation and transcriptional activation of genes involved in the T-cell activation prong of the immune response. Absent calcineurin activity, the T-cell activation cascade is interrupted because NFAT, in its phosphorylated state, cannot translocate to the cell nucleus.
Apart from its effects on the immune system, CsA has been shown to possess biological activity in the central nervous system. In rodent models of cerebral stroke, systemic treatment with CsA either before or following occlusion of the medial cerebral artery causes a reduction of infarct size [T. Yoshimoto and B. K. Siesjxc3x6, Brain Res., 839, pp. 283-91 (1999)]. CsA also protects against the decrease of acetyl choline receptors observed in the hippocampal formation after transient global forebrain ischemia [Y. Kondo et al., Neurochem Res., 24, pp. 9-13 (1999)], and has demonstrable neuroprotective effects in animal models of insulin-induced hypoglycemic coma [H. Friberg et al., J Neurosci., 18, pp. 5151-9 (1998)], traumatic brain injury [P. G. Sullivan et al., Exp Neurol., February 2000; 161, 631-7 (2000)], and experimental dopamine neuron degeneration [K. Matsuura et al., Exp. Neurol., 146, 526-351 (1997)]. In order for CsA to exert a protective effect after neural insult, it must be available at the site of injury. However, due to the blood-brain barrier, CsA shows only very limited penetration into the brain when administered systemically, and its best beneficial effects are seen if the blood-brain barrier is compromised [H. Uchino et al., Brain Res., 812, pp. 216-26 (1998); P. G. Sullivan et al., Exp. Neurol., 161, pp. 631-7 (2000)].
While the present invention is not bound by any particular theory, it appears that at least some of the effects of CsA on cells of the nervous system occur independently of calcineurin inhibition. Some of the inventors have previously shown that non-immunosuppressive peptidic analogues of CsA, which lack a calcineurin-binding domain, display neurotrophic activity in neural cell culture which is equal to that of CsA [J. P. Steiner et al., Nat. Med., 3, pp. 421-8 (1997)].
A number of types of mammalian cyclophilins have been identified and cloned, cyclophilins A, B, C, D, and cyclophilin-40 [Snyder and Sabatini, Nat. Med. 1:32-37 (1995); Friedman et al., Proc. Natl. Acad. Sci., 90:6815-6819 (1993)].
Cyclophilin B possesses an N-terminal signal sequence that directs translocation into the endoplasmic reticulum of the cell. The 23 kD cyclophilin C is found in the cytosol of the cell. Cyclophilin D, at 18 kD, appears to target its actions in the mitochondria, and cyclophilin-40 is a component of the inactivated form of a glucocorticoid receptor. Cyclophilin A is a 19 kD protein, which is abundantly expressed in a wide variety of cells. Like the other cyclophilins, cyclophilin A not only binds the immunosuppressive agent CsA, but it also possesses peptidyl-prolyl cis-trans isomerase (PPIase) and protein folding or xe2x80x9cchaperonexe2x80x9d activities. PPIase activity catalyzes the conversion of proline residues in a protein from the cis to the trans conformation [Fischer, et al., Biomed. Biochem. Acta 43:1101-1112 (1984)].
Since cyclophilin A was first identified as the receptor for CsA, the effects of the CsA:cyclophilin interaction have been well documented. Cyclosporin A binds to cyclophilin A with a dissociation constant in the range of 10xe2x88x928 mol/L, a value representing a relatively high degree of affinity [Handschumacher et al., Science 226:544 (1984)]. Knowledge about the interaction between drug and protein spawned a number of drug discovery efforts. Initially, the focus was on identifying novel immunosuppressive drugs that would mimic the effects of CsA without displaying its dose-limiting side-effects.
The field, however, lacks appreciation of the usefulness of cyclophilin-binding compounds for treating disease states, injuries and other abnormal conditions involving the central nervous system and other parts of the body. For therapeutic application in disorders of the central nervous system, for example, cyclophilin-binding compounds would need to penetrate from the bloodstream into the brain to bind to cyclophilin and exert biological effects. Cyclosporin A, however, generally displays poor penetration into the central nervous system after systemic administration, and therefore possess only low therapeutic potential for CNS applications if the blood-brain barrier is intact. See Uchino et al.; Sullivan et al., supra. Therefore, there exists need for safe and effective compositions and methodologies for treating disease states, injuries and other abnormal conditions involving the central nervous system and other organs by use of cyclophilin-binding compounds. These needs have gone unresolved until the development of the present inventions.
Researchers have also noted a functional association of cyclophilin A with the Gag protein of the HIV virus [Thali et al., Nature 372:363-365 (1994)]. This has taken drug development approaches in a new direction (See, for example, U.S. Pat. No. 5,767,069). Many researchers now seek to develop drugs that target the interaction between cyclophilin A and Gag in order to disrupt the HIV life cycle [Sternberg, BioWorld Today 7:1 (1996)].
The focus of the present invention is on non-peptidic small molecule compounds which interact with, have an affinity for, or bind to cyclophilin proteins. By studying the binding interaction of cyclophilin A and CsA, the inventors have designed and characterized a number of novel small molecule organic compounds which interact with cyclophilins, on the basis of which the inventors were able to develop and utilize screening procedures for rapidly identifying a class of similarly active compounds. These compounds have been specifically tested to show that they effect the growth and regeneration of cells of the nervous system, and protect such cells from otherwise lethal chemical injury. The compounds can be used in a number of ways, including therapeutic and research and development applications for various medical conditions, including neurological disorders.
The invention thus provides compounds that bind to CyP proteins. The compounds of this invention preferably do not suppress the immune system and preferably do not possess a biological activity involving binding to a FKBP, i.e., the compounds inhibit the peptidyl prolyl isomerase activity of FKBP with an IC50 of greater than 500 nM. A number of methods for determining the binding to CyPs and ways for exploiting the binding through in vitro and in vivo methods and uses are presented. Preferred compounds function to promote or affect neuronal cell growth or growth of nervous system cells, regenerate damaged or diseased neurons, or protect neurons or neuronal cells from death or degeneration following damage. Furthermore, aspects of this invention can be used in methods to identify and isolate additional CyP binding compounds or additional uses of the compounds.
The invention also provides a number of uses for these compounds, including uses that comprise the step of allowing the compound to contact an immunophilin protein. A variety of permutations of this method can be devised. In particular, the compounds can be used to affect the growth or resistance to noxious stimuli of neuronal cells, either in culture or in an animal. Thus, the compounds can be administered to cells or animals to affect a number of conditions associated with the decline, damage, or degeneration of nervous system cells or their physiological function.
In one aspect, the invention provides compounds of Formula I as shown and described below: 
where V is attached at position 4, 5, or 6, and is selected from the group consisting of C1-C4 alkyloxy, C1-C4 alkyl, C2-C4 alkenyloxy which is optionally substituted with Q, C1-C4 alkoxycarbonyl, Cl-C6 alkylcarbamoyl which is optionally substituted with amino or C1-C4 alkoxycarbonylamino; di-(C1-C4 alkyl)carbamoyl, C1-C4 alkanoyl, Q-substituted C1-C6 alkyl, alkenyl, or alkynyl, COxe2x80x94W, and xe2x80x94(CH2)nxe2x80x94W;
wherein W is Q, xe2x80x94Zxe2x80x2xe2x80x94(CH2)mxe2x80x94Q, xe2x80x94Nxe2x95x90CHxe2x80x94(CH2)mxe2x80x94Q, COOCH3, COCH3, hydroxyl, mercaptyl, amino, nitro, halo, carboxy, trifluoromethyl, or C6 alkylamino substituted with amino or C1-C4 alkoxycarbonylamino;
n and m are independently 0-4;
nxe2x80x2 is 0-3;
Zxe2x80x2 is O, S, NH, or NR;
where X and Y are the same or different, and may independently be: 
xe2x80x83and where Y may further be: Q, 
xe2x80x83or C1-C6 straight or branched chain alkyl, alkenyl, or alkynyl which is substituted at one or several positions with Q, and which further is optionally substituted at one or several positions by hydroxyl, mercaptyl, or carbonyl oxygen;
wherein Z is O or S;
and wherein R may independently be:
Q,
or C1-C6 straight or branched chain lower alkyl, alkenyl or alkynyl which is substituted at one or several positions with Q, and which further is optionally substituted in one or several positions by hydroxyl, mercaptyl, or carbonyl oxygen, and wherein one or more of the carbon atoms are optionally replaced with O, N, NH, S, SO, or SO2;
and wherein Q is a mono-, bi-, or tricyclic, carbo- or heterocyclic ring which is saturated, partially saturated, or aromatic, and wherein the individual ring sizes are 5-6 members, and wherein each heterocyclic ring, if present, contains 1-4 heteroatoms independently selected from the group consisting of O, N, and S in any chemically stable order and oxidation state, and wherein Q is optionally substituted in one or several positions with:
halo; hydroxyl; mercaptyl; nitro; trifluoromethyl; aminocarbonyl; arylaminocarbonyl in which the aryl is optionally halogenated and optionally substituted with trifluoromethyl or cyano; C1-C4 alkylsulfonyl; C1-C4 alklylthio; oxo; cyano; carboxy; C1-C6 alkyl or alkenyl; C1-C4 alkoxy; C1-C5 alkoxycarbonyl; C1-C4 alkenyloxy; phenoxy; phenyl; cyanophenyl; benzyloxy; benzyl; amino; C1-C4 alkylamino; di-(C1-C4) alkylamino; C1-C4 alkylcarbamoyl; di(C1-C4)alkylcarbamoyl;
or a combination thereof;
provided that:
when X and Y are 
xe2x80x83or a combination thereof, and
nxe2x80x2 is 0, and
n is 0, and
V is halo, hydroxyl, nitro, trifluoromethyl, C1-C4 alkoxy orxe2x80x94alkenyloxy, phenoxy, benzyloxy, amino, or Q,
then R is not Q, or C1-C3 branched or straight chain alkyl substituted with Q.
Preferred compounds under this aspect of the invention are substituted with V at position 5. Preferably, V is xe2x80x94(CH2)nxe2x80x94Zxe2x80x2xe2x80x94(CH2)mxe2x80x94Q, or Q-substituted C1-C6 straight or branched chain alkyl or alkenyl.
In a further group of preferred compounds nxe2x80x2 is 0, and X and Y are independently 
The compounds of this invention can be selected for use from Formula I. Starting with a particular compound, any of the individual variable groups R, X, Y, Z, Zxe2x80x2, Q, V, and a value for n, nxe2x80x2 and m can be selected while one or more of the other variable groups can be modified. For example, in Formula I, V can be attached at position 5 and specified to be xe2x80x94(CH2)nxe2x80x94NHxe2x80x94(CH2)mxe2x80x94Q to select subgroups of compounds which share a common 1,3,5,-substitution pattern wherein at least one of the Q groups is attached via an amine linkage.
Any of the subgroups thus obtained can be further divided into additional subgroups of compounds defined by the allowed combinations of X and Y, and by requiring that X and Y are either similar, or different from each other, and by requiring, for example, that R be Q, or that R be Q-substituted alkyl, alkenyl, or alkynyl, and that all Q-substituents be the same, or different from each other. This process can be repeated using any one, or a combination of, the variable groups. In this way, one skilled in the art can select and use groups of related compounds or even individual compounds, all within the invention. Many examples are shown below; however, they are merely representative of the scope of changes and modifications possible. One skilled in the art can devise many separate compounds from the description of Formula I alone.
In this specification, the generic terms xe2x80x9calkylxe2x80x9d, xe2x80x9calkenylxe2x80x9d or xe2x80x9calkynylxe2x80x9d include both straight-chain and branched-chain saturated or unsaturated groups. xe2x80x9cArylxe2x80x9d in terms such as xe2x80x9carylaminocarbonylxe2x80x9d typically means groups such as phenyl, naphthyl, pyrrolyl, pyrrolidinyl, pyridinyl, pyrimidinyl, purinyl, furyl, imidazolyl, quinolinyl, oxazolyl, thiazolyl, pyrazolyl, and thienyl. xe2x80x9cAlkyloxyxe2x80x9d or xe2x80x9calkoxyxe2x80x9d refer to groups such as, for example, methoxy or ethoxy; xe2x80x9calkoxycarbonylxe2x80x9d refers to groups such as, for example, methyl ester or butyl ester; xe2x80x9cC1-C6 alkylcarbamoylxe2x80x9d and xe2x80x9cdi(C1-C4)alkylcarbamoylxe2x80x9d refer to saturated or unsaturated carbon chains attached via an amide linkage, such as, for example, ethylcarbamoyl or diethylcarbamoyl; xe2x80x9cC1-C4 alkoxycarbonylaminoxe2x80x9d refers to groups such as, for example, tert-butoxycarbonylamino (C4H9)xe2x80x94Oxe2x80x94COxe2x80x94NHxe2x80x94; xe2x80x9carylaminocarbonylxe2x80x9d refers to groups such as phenylaminocarbonyl (C6H5)xe2x80x94NHxe2x80x94COxe2x80x94; xe2x80x9cC1-C4 alkanoylxe2x80x9d refers to groups such as, for example, formyl or acetyl; xe2x80x9cC6 alkylaminoxe2x80x9d refers to groups such as, for example, hexylamino.
Compounds of Formula I may be prepared or formulated as a salt or derivative for some uses, including pharmaceutical and tissue or cell culture uses. As used herein, the CyP-binding compounds of this invention are defined to include pharmaceutically acceptable derivatives. A xe2x80x9cpharmaceutically acceptable derivativexe2x80x9d denotes any pharmaceutically acceptable salt, ester, thioester, or salt of such ester or thioester, of a compound of this invention or any other compound which, upon administration to an animal or human patient, is capable of providing (directly or indirectly) a compound of this invention, or a metabolite or residue thereof, characterized by the ability to bind to a CyP and/or its usefulness in treating or preventing a medical disorder. Examples of medical disorders within the scope of this aspect of the invention are given below. The compounds of the invention can also be part of a composition comprising one or more compounds of Formula I.
The compounds of the invention can be produced as a mixture of isomers or racemic mixtures or as optically pure compounds. Methods for separating stereoisomers known in the art can also be used to enrich mixtures for one or more compounds. The compositions of the invention may similarly contain mixtures of stereoisomers, mixtures of one or more stereoisomers, or be enriched for one or more stereoisomers. All of these forms are specifically included in this invention and are intended to be included in the claims.
Preferably, compounds of Formula I selectively bind to a CyP as detected, for example, by a measurable inhibition of the peptidyl-prolyl cis-trans isomerase enzyme activity (PPIase) of CyP. xe2x80x9cSelectively bind to a CyPxe2x80x9d means the compounds do not possess a significant binding affinity toward a FKBP and/or do not possess a biological activity associated with binding to a FKBP. For example, the IC50 towards FKBP is at or above 500 nM. The skilled artisan is familiar with ways to detect rotamase inhibition in CyP and FKBP. In addition, a number of ways for detecting binding to a CyP are described below.
As is readily apparent from Formula I, a common substitution pattern exists, wherein at least two carbo- or heterocyclic groups are attached to a central trisubstituted phenyl ring by a combination of straight or branched linker chains. This common pattern differs from the approaches previously taken to identify other immunophilin binding compounds or drugs. For example, Holt et al. [Bioorg. Med. Chem. Letters, 4: 315-320 (1994)] discuss a pipecolate, or 1-(1,2-dioxo)2-carboxylate piperidine containing base structure for binding to FKBP. Similarly, earlier work by the inventors established the relevance of a 1-(1,2-dioxo)2-carboxylate pyrrolidine containing structure for binding to FKBP [Steiner et al., Proc. Natl. Acad. Sci. U.S.A. 94:2019-2024 (1997)]. Presumably, these structures mimic the natural substrate for the peptidyl-prolyl-isomerase (PPIase) activity, a proline-containing fragment of a protein. In a protein, the amino acid proline corresponds to a 1,2-substituted pyrrolidine structure. Prior work has generally incorporated that structure. However, Formula I does not correspond to a 1,2-substituted pyrrolidine structure. Yet, as demonstrated here, compounds of this formula possess important bioactive and biochemical functions.
The body of work related to analogues of cyclosporin A, FK-506, and rapamycin further distances the compounds of this invention from prior work. (See, for example, U.S. Pat. Nos. 5,767,069, 5,284,826, 4,703,033, and 5,122,511). These analogues typically possess a cyclic peptide structure.
In another aspect, the invention relates to methods for binding non-peptidic compounds to cyclophilin-type immunophilins. While the present invention is not bound by this theory, it is hypothesized that binding results in an xe2x80x9cimmunophilin:drugxe2x80x9d complex, which is considered to be the active agent in the in vivo immunosuppressive and neurotrophic activities of PPIase inhibitors [Hamilton and Steiner, J. Med. Chem. 41:5119-5143 (1998); Gold, Mol. Neurobiol. 15:285-306 (1997)]. Whether or not the complex acts for any or all the therapeutic actions of these PPIase inhibitors, focusing on the immunophilin:drug interaction has led to the discovery a number of new drug compounds. Accordingly, methods of using compounds, such as those of Formula I, to create an immunophilin:compound complex, or a CyP:compound complex, provide an important aspect of this invention. This aspect can be exploited, for example, in methods where the compound, or a mixture comprising one or more of the compounds of the invention, or a pharmaceutical composition comprising one or more of the compounds of the invention, is administered to cells in culture or to an animal.
While the immunophilin:compound complex has beneficial effects in vivo and in vitro in cultured cells, numerous other uses for binding the compounds to an immunophilin exist. For example, further in vitro binding experiments can be used to identify and purify cellular components that interact with the immunophilin complex in a cell-free environment, as would be the case where an affinity chromatography column or matrix bearing the compound is reacted with a CyP, and cellular or tissue extracts containing a CyP are passed over the column or matrix.
Thus, the invention also provides methods for forming immunophilin:compound or CyP:compound complexes as well as the complexes themselves. To form these complexes, the compounds can contact an immunophilin or CyP protein in vivo, in cell or tissue culture, or in a cell-free preparation. In preferred embodiments, the compound contacts a human CyP protein, such as one or more of CyP A, B, C, D, or xe2x88x9240. The CyP protein can be native to the cell or organism, produced via recombinant DNA, produced by other manipulations involving introduced genetic material, or produced by synthetic means. Furthermore, chimeric proteins possessing immunophilin domains that function to bind immunophilin ligands can also be used to form a protein:compound complex. The formation of the CyP:compound, immunophilin:compound, or protein:compound complex need not be irreversible.
The binding of a compound to a CyP can be detected in a number of ways, including PPIase inhibition assay, affinity chromatography, in vivo neuroprotection or neuroregeneration activity assay, in vitro neurotrophic activity assay, or by any of the activities in neuronal cells or cells of the nervous system described below, in the examples, or in the cited references.
The invention also provides compositions comprising at least one compound of Formula I. The compositions may comprise one or more pharmaceutically acceptable carriers, excipients, or diluents. These compositions, or the compounds themselves, or mixtures of said compounds or compositions, can be administered to an animal. Administration can be one method to allow the compound to contact a CyP within the animal. As one skilled in the art would recognize, various routes of administration are possible. Exemplary routes are specifically described in the detailed description below.
The compounds of Formula I, or compositions comprising them, can function to regenerate nerve cells, promote neurite outgrowth, and protect nerves from otherwise damaging treatments or conditions. Thus, the compounds and compositions of this invention are useful in the diagnosis, cure, mitigation, treatment, or prevention of neurological conditions in animals, including humans, and in animals (including humans) exposed to neurodegenerative agents or having damaged nervous system cells. Such conditions and disorders, when present in an animal, including humans, can be neurodegenerative disorders, neuropathic disorders, neurovascular disorders, traumatic injury of the brain, spinal cord, or peripheral nervous system, demyelinating disease of the central or peripheral nervous system, metabolic or hereditary metabolic disorder of the central or peripheral nervous system, or toxin-induced- or nutritionally related disorder of the central or peripheral nervous system. When present in a human, a neurodegenerative disorder can be, for example, Parkinson""s disease, Alzheimer""s disease, amyotrophic lateral sclerosis (ALS), Huntington""s disease, cerebellar ataxia, or multisystem atrophy including, for example, olivopontocerebellar degeneration, striatonigral degeneration, progressive supranuclear palsy, Shy-Drager syndrome, spinocerebellar degeneration and corticobasal degeneration. A demyelinating disease can be, for example, multiple sclerosis, Guillain-Barrxc3xa9 syndrome, or chronic inflammatory demyelinating polyradiculoneuropathy. A neurovascular disorder can be global cerebral ischemia, spinal cord ischemia, ischemic stroke, cardiogenic cerebral embolism, hemorrhagic stroke, lacunar infarction, multiple infarct syndromes including multiple infarct dementia, or any disorder resulting in ischemia or ischemia/reperfusion injury of the central nervous system. Traumatic injury of the central or peripheral nervous system can be, for example, concussion, contusion, diffuse axonal injury, edema, and hematoma associated with craniocerebral or spinal trauma, or axonal or nerve sheath damage associated with laceration, compression, stretch, or avulsion of peripheral nerves or plexi, and further includes nerve damage caused during surgery, such as prostate surgery. A neuropathic disorder can be, for example, diabetic neuropathy, uremic neuropathy, neuropathy related to therapy with drugs such as phenytoin, suramin, taxol, thalidomide, vincristine or vinblastine; or neuropathy/encephalopathy associated with infectious disease, such as, for example, encephalopathy related to HIV, rubella virus, Epstein-Barr virus, herpes simplex virus, toxoplasmosis, prion infection. A metabolic disorder of the central nervous system can be, for example, status epilepticus, hypoglycemic coma, or Wilson""s disease.
The following detailed description should not be taken as a limitation on the scope of the invention, and all embodiments and examples given are merely illustrative of the invention. Additional aspects of the invention can be devised by reference to this disclosure as a whole in combination with the references cited and listed throughout and at the end of the specification and the knowledge of one skilled in the art. All of the references cited and listed can be relied on, in their entirety, to allow one to make and use these additional aspects of the invention.
One skilled in the art can refer to general reference texts for detailed descriptions of known techniques discussed herein or equivalent techniques. These texts include Current Protocols in Molecular Biology [Ausubel, et al., eds., John Wiley and Sons, N.Y., and supplements through June 1999), Current Protocols in Immunology (Coligan, et al., eds., John Wiley and Sons, N.Y., and supplements through June 1999)], and Current Protocols in Pharmacology (Enna et al., eds., John Wiley and Sons, N.Y., and supplements through June 1999) for example, each of which are specifically incorporated by reference in their entirety. These texts can also be referred to in making or using an aspect of the invention.
As noted above, cyclosporin A was the first compound identified to bind a CyP. Based on the cyclic structure of cyclosporin A, a number of large, usually cyclic peptides were developed as immunosuppressive compounds that bind CyP. Now, unexpectedly, the inventors have found a non-peptidic class of CyP binding compounds with activity in neuronal cells. The following compounds are representative of those tested. 
The compounds of the invention can be prepared by a number of synthetic routes. The examples below detail schemes I to XIV and the preparation of specific compounds. However, one skilled in the art can modify the steps, reactants, and reaction conditions in the examples and schemes to arrive at numerous examples of compounds of the invention. In addition, if particular stereoisomers or mixtures are desired, the starting materials and/or reactants in the preparatory scheme can be selected and used accordingly. Alternatively or in addition, particular intermediates can be purified or enriched by chromatographic or enzymatic methods, or by manipulating reaction conditions or selective crystallization, to generate particular final products or mixtures. One skilled in the art is familiar with numerous methods to selectively produce or enrich for desired stereoisomers or mixtures. All of the compounds of the examples, including the intermediates, are specifically included in the compounds of the invention and can be used in the methods of the invention. Specific examples of synthetic intermediates which are useful as compounds of this invention include N-(3-amino-5-{[3-(trifluoromethyl)phenyl]amino}phenyl)(3,5-dichlorophenyl)formamide, used in the preparation of Exemplary Compound 10; N-(5-(2-aza-3-naphthylprop-2-enyl)-3)-{[3-(trifluoromethyl)phenyl]carbonylamino}phenyl)[3-(trifluoromethyl)phenyl]formamide, used in the preparation of Exemplary Compound 14; (3-bromo-5-{N-[3-(trifluoromethyl)phenyl]carbamoyl}phenyl)-N-[3-(trifluoromethyl)phenyl]formamide, used in the preparation of Exemplary Compounds 23-25; 1-{3-[(2-Naphthyl)methoxy]-5-nitrobenzoyl}-2-benzoylhydrazine, used in the preparation of Exemplary Compound 15; and 3,5-bis(benzyloxy)benzoate; 3,5-Bis(Benzyloxy)benzoic acid; and 3,5-Bis(Benzyloxy)benzoic acid chloride, used in the synthesis of Exemplary Compound 8.
The compounds of the invention may be prepared as salts or derivatives. Various salts and derivatives are known in the art and a non-limiting list of possible choices includes acid salts: acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, mesylate, dimesylate, palmoate, pectinate, persulfate, 3-phenylpropionate, phosphates, picrate, pivalate, propionate, succinate, sulfates, tartrate, thiocyanate, tosylate, and undecanoate. Base salts may include: amine salts, ammonium salts, alkali metal salts such as sodium and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, salts with organic bases such as dicyclohexylamine salts, N-methyl-D-glucosamine, and salts with amino acids, for example arginine or lysine. Nitrogen-containing groups of the compound can be quatemized with agents as: alkyl halides, for example methyl, ethyl, propyl, and butyl chlorides, bromides, or iodides; dialkyl sulfates, for example dimethyl, diethyl, dibutyl and diamyl sulfates, long chain halides, for example decyl, dodecly, lauryl, myristyl, or stearyl chlorides, bromides, or iodides; and aralkyl halides, for example benzyl and phenethyl bromides, chlorides, or iodides. The skilled artisan is familiar with methods for producing and testing any suitable salt or derivative. (See, for example, Remington""s Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 18th Edition, specifically incorporated herein by reference.)
In general, activity in the nervous system for a particular compound can be identified by assaying for the ability to promote neurite outgrowth, protect neurons from damage by chemical treatments, promote the growth of neurons or neuronal cells, recover lost or damaged motor, functional or cognitive ability associated with nervous tissue or organs of the nervous system, or regenerate neurons. These activities can be useful in treating, diagnosing, or prognosing a number of human disease conditions, including, but not limited to, the neurological conditions described above, as well as disorders of the retina and optic nerve, vestibulocochlear disorders, and erectile dysfunction related to nerve damage caused during prostate surgery.
A number of animal model assays and cell culture assays have been developed and can be relied on for their clinical relevance to disease treatments, including the human diseases noted above. Each of the following references can be used as a source for these assays, and all of them are specifically incorporated herein by reference in their entirety for that purpose: Steiner, et al., PNAS 94: 2019-2024 (1997); Hamilton, et al., Bioorgan. Med. Chem. Lett. 7:1785-1790 (1997); McMahon, et al., Curr. Opin. Neurobiol. 5:616-624 (1995); Gash, et al., Nature 380:252-255 (1996); Gerlach, et al., Eur. J. Pharmacol.-Mol. Pharmacol. 208:273-286 (1991); Apfel, et al., Brain Res. 634:7-12 (1994); Wang, et al., J. Pharmacol. Exp. Therap. 282:1084-1093 (1997); Gold, et al., Exp. Neurol. 147:269-278 (1997); Hoffer et al., J. Neural Transm. [Suppl.] 49:1-10 (1997); Lyons, et al., PNAS 91:3191-3195 (1994); Yoshimoto and Siesjxc3x6, Brain Res., 839, pp. 283-91 (1999); Kondo et al., Neurochem Res., 24, pp. 9-13 (1999); Friberg et al., J Neurosci., 18, pp. 5151-9 (1998); Sullivan et al., Exp Neurol., February 2000; 161, 631-7 (2000).
Preferred methods for detecting neuronal activity include a neuroprotective assay, for example an organotypic slice culture of the spinal cord, in which a compound is tested for the ability to protect against treatment causing glutamate neurotoxicity. Sensory neuronal cultures of the dorsal root ganglia (DRG) can also be assayed for neurite outgrowth, an assay for neurotrophic activity. Cultured cells are treated with a compound of the invention and later assayed for the presence of new neurite fibers. The compounds can also be tested for their ability to inhibit the mitochondrial permeability transition by measuring large amplitude mitochondrial swelling of freshly isolated rat liver mitochondria in a spectrophotometric assay [Broekemeier, et al., J. Biol. Chem. 264: 7826-7830 (1989)].
The compounds of the present invention can further be assayed for their in vivo potency and efficacy using a common mouse model of a neurodegenerative disorder: Mice can be treated orally or subcutaneously, for example, with the compounds of the present invention, and subsequently be subjected to MPTP-treatment. MPTP (N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) is a systemically available neurotoxin that selectively destroys the dopaminergic neurons of the ventral midbrain as well as their forebrain projections. One skilled in the art is familiar with methods for assessing the integrity of the midbrain-forebrain projection in MPTP-lesioned mice that were treated with the compounds of this invention, and a relative preservation of the nerve fibres, nerve terminals, or of the dopaminergic cell bodies in the ventral midbrain, would be indicative of the relative neuroprotective efficacy of the compounds of this invention.
In the assays here exemplified, immunohistochemistry can aid in the visualization and quantitation of neurites, terminals and cell bodies.
The compounds of the invention can also be used to promote the establishment or maintenance of tissue or cell cultures. Similar to the use for promoting neuronal cell growth, the compounds can be added to primary, transformed, or established cell cultures. Particularly in the case of neuronal cells, the compounds can induce growth in culture and extend the culture lifetime of cells.
A recognized method for assessing the affinity of the compound to cyclophilin is the rotamase inhibition assay. For this purpose, the following references are specifically incorporated by reference and can be relied on to make assays of rotamase inhibition: Fischer, et al., Biomed. Biochem. Acta 43:1101-1112 (1984); Kofron, et al., Biochem. 30:6127-6134 (1991); Kofron et al., J. Am. Chem. Soc. 114:2670-2675 (1992); Harrison et al., Biochem. 29:3813-3816 (1990); Lang et al., Nature 329:268-270 (1987); Mucke et al., Biochem. 31:7848-7854 (1992); Schonbrunner et al., J. Biol. Chem. 266:3630-3635 (1991); Hsu et al., J. Am. Chem. Soc. 112:6745-6747 (1990); and Justice et al., Biochem. Biophys. Res. Commun. 171:445-450 (1990).
Additional uses for the compounds, which may or may not relate to CyP binding, are also included in the methods of the invention. For example, the compounds are useful to promote hair growth, and to prevent or retard hair loss. In murine models which mimic human premature hair follicle regression or human chemotherapy-induced hair loss, topical application of CsA was found to induce and maintain hair growth, and topical or systemic administration of CsA was found to protect from hair loss induced by cancer chemotherapeutic agents [see, e.g., Maurer, et al. Am. J. Pathol. 150 (4):1433-41 (1997); Paus, et al., Am. J. Pathol. 144, 719-34 (1994)]. It has been speculated that initiation of hair growth by CsA is unrelated to immunosuppression [Iwabuchi, et al., J. Dermatol. Sci. 9, 64-69 (1995)]. The compounds of the invention are useful in preventing or retarding hair loss in patients undergoing therapy with doxorubicin, carboplatin, cisplatin, cyclophosphamide, dactinomycin, etoposide, hexamethamelamine, ifosfamide, taxol, vincristine, bleomycin, 5-fluorouracil, and other agents useful in the therapy of cancer. The compounds of the invention are further useful in promoting hair growth in patients suffering from hair loss caused by one or a combination of the aforementioned chemotherapeutic agents. The compounds of the invention are further useful in the prevention of hair loss, and in the promotion of hair growth, in patients undergoing radiation therapy, and in patients suffering from alopecia areata, androgenetic alopecia/male pattern baldness, anagen effluvium, trichotillomania, traction alopecia, telogen effluvium, and hair loss induced by drugs such as, for example, methotrexate, nonsteroidal anti-inflammatory drugs, or beta blockers.
For these purposes, the compounds may be administered as part of pharmaceutical or cosmetic compositions, singly, in combination with other compounds of the invention, in combination with other hair growth-promoting or hair-loss preventing agents, or in combination with one or several other active agents such as, for example, antibiotic agents, antidandruff agents, and anti-inflammatory agents.
The compounds of the invention are also useful to treat or effect mitochondrial disorders, metabolic disorders, diabetes, or vision loss. The mitochondrion is increasingly being recognized as an important mediator of cell death in hypoxia, ischemia, and chemical toxicity. Disruption of the mitochondrial transmembrane potential is observed before other features of apoptosis (e.g. generation of reactive oxygen species or internucleosomal DNA fragmentation (xe2x80x9cladderingxe2x80x9d)) become detectable. This applies to many different models of apoptosis induction, such as, for example, NGF-deprivation of cultured sympathetic neurons, dexamethasone-induced lymphocyte apoptosis, programmed lymphocyte death, activation-induced programmed cell death of T cell hybridomas, and tumor necrosis factor-induced death of lymphoma cells. [Marchetti, P., et al., J. Exp. Med. 184 (1996) 1155-1160]. Breakdown of mitochondrial transmembrane potential in proapoptotic cells has been attributed to the formation of an unspecific high conductance channelxe2x80x94the mitochondrial permeability transition porexe2x80x94which leads to an increased permeability of the inner mitochondrial membrane to small molecular weight solutes. The ensuing release of intramitochondrial ions, influx of solutes, uncoupling of oxidative phosphorylation, and loss of metabolic intermediates accompanies large amplitude mitochondrial swelling and a depletion of cellular energy stores [see, e.g., Lemasters, J. J. et al., Mol. Cell. Biochem. 174 (1997) 159-165]. Importantly, CsA and non-immunosuppressive peptidic CsA analogues have been described to potently block pore conductance and inhibit the onset of the mitochondrial permeability transition [Broekemeier, K. M., et al., J. Biol. Chem. 264 (1989) 7826-7830; Zamzami, M., et al., FEBS Lett. 384 (1996) 53-7]. The mitochondrial permeability transition pore forms under calcium overload conditions such as occur in ischemia/reperfusion injury, and it has been found that administration of CsA and/or non-immunosuppressive peptidic CsA analogues, by blocking the permeability transition pore, leads to significant protection in experimental models of cerebral stroke [Matsumoto, S., et al., J. Cereb. Blood Flow Metab. 19 (1999) 736-41], cardiac ischemia [Griffiths, E. J. and Halestrap, A. P., J. Mol. Cell Cardiol. 25 (1993) 1461-1469], and hepatic ischemia/reperfusion injury [Leducq, N., et al., Biochem. J. 336 (1998) 501-6]. The compounds of the invention are useful in blocking the mitochondrial permeability transition pore; inhibiting breakdown of mitochondrial metabolism in cells which undergo oxidative stress, calcium overload, excitotoxic or hypoglycemic injury both in vitro and in vivo; inhibiting mitochondrial swelling; inhibiting, both in vivo and in vitro, breakdown of energy metabolism and cell death of mammalian cells following either physiological induction of programmed cell death through signal molecules such as, for example, tumor necrosis factor, or following physiological stress related to hypoxia, hypoglycemia, excitotoxic insult, or calcium overload. The inventive compounds are useful in preventing or delaying cell death in large scale/commercial scale cell culture. The compounds of the invention are further useful in the diagnosis, cure, mitigation, treatment, or prevention of ischemic injury or ischemia/reperfusion injury, such as mesenteric infarction, bowel ischemia, hepatic infarction or ischemia/reperfusion injury, renal infarction, splenic infarction, or cardiac ischemia or ischemia/reperfusion injury related, for example, to angina pectoris, congestive heart failure, or myocardial infarction. Additional uses of the compounds of the invention include applications in the diagnosis, cure, mitigation, treatment, or prevention of Reye""s syndrome; ophthalmic disorders such as glaucoma, ischemic or vascular retinopathies, or degeneration of the photoreceptor cell layer. The invention also provides a method of preventing or reducing tissue damage of organs used in organ transplantation surgery, comprising contacting said organs with a compound of Formula I.
CsA and its non-immunosuppressive peptidic analogues have also been found to potently inhibit the growth of pathogenic protozoan parasites, such as Cryptosporidium parvum, Plasmodium falciparum, Plasmodium vivax, Schistosoma spec., and Toxoplasma gondii [Perkins, et al., Antimicrob. Agents Chemother. 42: 843-848 (1998)]. Although antiprotozoan activity appears not to be correlated with immunosuppressive or PPIase inhibitory activity [Bell, et al., Biochem. Pharmacol. 48:495-503 (1994); Khattab, et al., Exp. Parasitol. 90:103-109 (1998)], the protozoan cyclophilin, complexed to CsA or its noni-mmunosuppressive analogues, has been proposed to play an active role in mediating the antiparasitic effects of peptidic cyclophilin ligands [Berriman and Fairlamb, Biochem. J. 334:437-445 (1998)]. CyA and its non-immunosuppressive analogues also inhibit reproduction of filarial parasites in vivo with a potency unrelated to their immunosuppressive activity and their activity against Plasmodium [Zahner and Schultheiss, J. Helminthol. 61:282-90 (1987)], and have been shown to exert direct antihelmintic effects [McLauchlan, et al., Parasitology 121:661-70 (2000)].
The compounds of this invention are useful in the diagnosis, cure, mitigation, treatment, or prevention of infections with pathogenic protozoan or helmintic parasites in animals, including humans. In humans, the present compounds find application in the treatment of conditions such as, for example, malaria, river blindness, lymphatic filariasis, intestinal roundworm infection, tapeworm infection, pinworm infection, toxoplasmosis, leishmaniasis, trypanosomiasis, and bilharzia.
The compounds of this invention are also useful in affecting the viral replication process of the HIV-1 virus. The infectivity of the HIV-1 virus is believed to depend critically upon an interaction of the viral Gag polyprotein capsid complex with host Cyclophilin A. [Streblow et al. Virology 245 (1998) 197-202; Li et al. J. Med. Chem. 43, (2000) 1770-9]. The compounds of this invention can function to inhibit or disrupt the interaction of human host CyPA with HIV-1 Gag proteins, to decrease or eliminate the infectivity of the HIV-1 virus, to treat or prevent infection of humans with the HIV-1 virus, and to treat or prevent acquired immune deficiency syndrome (AIDS) associated with HIV-1 infection. The compounds of this invention are further useful in the diagnosis, treatment, cure, mitigation or prevention of infections with strains of the human immunodeficiency virus other than HIV-1, and of infections caused by other pathogenic viruses, such as influenza viruses.
The compounds of the invention have utility in pharmacological compositions for the treatment and prevention of various neurological, ischemic, and inflammatory disorders or for various in vitro and cell culture treatments. The compounds also have utility in pharmacological compositions for the treatment and prevention of HIV-infection, promotion of hair growth, immunosuppression, mitochondrial disorders, traumatic injury to nervous tissue, or conditions associated with retinal and optic nerve damage. The compounds of the invention may be prepared as a salt or derivative, as described above.
A compound of the invention can be administered to an animal or human patient by itself or in pharmaceutical compositions where it is mixed with suitable carriers or excipients, at doses to treat or ameliorate various conditions. The compounds according to the present invention preferably have sufficient stability, potency, selectivity, solubility and availability to be safe and effective in treating diseases, injuries and other abnormal conditions or insults to the central nervous system, the peripheral nerves, and other organs. A therapeutically effective dose refers to that amount of the compound sufficient to effect an activity in a nerve or neuronal cell, to produce a detectable change in a cell or organism, or to treat a disorder in a human or other mammal. The word xe2x80x9ctreatxe2x80x9d in its various grammatical forms as used in relation to the present invention refers to preventing, curing, reversing, attenuating, alleviating, minimizing, suppressing, ameliorating or halting the deleterious effects of a disease state, disease progression, injury, wound, ischemia, disease causative agent (e.g., bacteria, protozoans, parasites, fungi, viruses, viroids and/or prions), surgical procedure or other abnormal or detrimental condition (all of which are collectively referred to as xe2x80x9cdisorders,xe2x80x9d as will be appreciated by the person of skill in the art). A xe2x80x9ctherapeutically effective amountxe2x80x9d of a compound according to the invention is an amount that can achieve effective treatment, and such amounts can be determined in accordance with the present teachings by one skilled in the art.
The methods of the present invention comprise (i.) administration of a compound of Formula I, where the compound is itself therapeutically active in the treatment of the targeted medical condition, or (ii.) administration of a prodrug of a compound of Formula I, wherein such prodrug is any compound which is capable of undergoing metabolic conversion to a compound of Formula I following administration, or (iii.) administration of a compound of Formula I where the compound is capable of undergoing metabolic conversion to a metabolite following administration, and where the metabolite is therapeutically active in the treatment of the targeted medical condition, or (iv.) administration of a metabolite of a compound of Formula I, where the metabolite is therapeutically active in the treatment of the targeted medical condition. Thus, the use of a compound of Formula I in the methods of the present invention explicitly includes not only the use of the compound itself, but also the modifications ii, iii, and iv discussed in this paragraph, and all such modifications are explicitly intended to be within the scope of the following claims.
Therapeutically effective doses may be administered alone or as adjunctive therapy in combination with other treatments. Techniques for the formulation and administration of the compounds of the instant application may be found in Remington""s Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 18th edition (1990).
Suitable routes of administration may, for example, include oral, rectal, transmucosal, buccal, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections, and optionally in a depot or sustained release formulation. Furthermore, one may administer the agent of the present invention in a targeted drug delivery system, for example in a liposome coated with an antibody. The liposomes will be targeted to and taken up selectively by cells expressing the appropriate antigen.
The pharmaceutical compositions of the present invention may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, emulsifying, encapsulating, entrapping, or lyophilizing processes. Pharmaceutical compositions for use in accordance with the present invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active compounds into preparations, which can thus be used pharmaceutically.
For injection, the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers, such as Hank""s solution, Ringer""s solution, or physiological saline buffer. For transmucosal or buccal administration, penetrants appropriate to the barrier to be permeated may be used in the formulation. Such penetrants are known in the art.
For oral administration, the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers, well known to those in the art. Such carriers enable the compounds of the invention to be formulated as tablets, pills, capsules, liquids, quick-dissolving preparations, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated. Pharmaceutical preparations for oral use of the compounds of this invention can be obtained by employing a solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP).
In general, the pharmaceutical compositions also may comprise suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols. If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate or a number of others disintegrants [see, for example, Remington""s Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 18th edition (1990)].
For administration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, pressurized air, or other suitable gas or mixture. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g. gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
The compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents, which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. Alternatively, the active ingredient may be in powder form for reconstitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
The compounds may also be formulated in rectal compositions such as suppositories, e.g., containing conventional suppository bases such as cocoa butter or other glycerides. In addition to the formulations described previously, the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
The compounds of the invention may further be formulated in pharmaceutical or cosmetic compositions for topical application to the skin in the form of an aqueous, alcoholic, aqueous/alcoholic or oily solution, or of a dispersion of the lotion or serum type, of an emulsion having a liquid or semi-liquid consistency of the milk type, obtained by dispersion of a fatty phase in an aqueous phase (O/W) or vice versa (W/O), or of a suspension or of an emulsion with a soft consistency of the aqueous or anhydrous gel, foam or cream type, or, alternatively, of microcapsules or microparticles, or of a vesicular dispersion of ionic and/or nonionic type, or may further be administered in the form of an aerosol composition comprising a pressurized propellent agent. The compounds of the invention can also be formulated into various compositions for hair care and, in particular, shampoos, hair-setting lotions, treating lotions, styling creams or gels, dye compositions (in particular oxidation dyes), optionally in the form of color-enhancing shampoos, hair-restructuring lotions, permanent-wave compositions, and the like. Pharmaceutical or cosmetic compositions comprising compounds of the invention can also contain additives and adjuvants which are conventional in the cosmetics field, such as gelling agents, preservatives, antioxidants, solvents, fragrances, fillers, screening agents, odor absorbers and colorants. The amounts of these different additives and adjuvants are those typically employed in the cosmetics field and range, for example, from 0.01% to 20% of the total weight of the composition, preferably 0.1% to 10%, and more preferably 0.5% to 5%. In addition to one or several compounds of the invention, compositions for topical application may further contain additional agents already known in the art to promote hair growth or to prevent or retard hair loss, such as, without limitation, tocopherol nicotinate, benzyl nicotinate or 2,4-diamino-6-piperidinopyrimidine 3-oxide, or may contain other active agents such as antibacterial agents, antiparasitic agents, antifungal agents, antiviral agents, anti-inflammatory agents, antipruriginous agents, anaesthetic agents, keratolytic agents, antiseborrhoeic agents, antidandruff agents, or antiacne agents. The cosmetic or pharmaceutical compositions according to the invention can be topically applied onto the alopecic areas of the scalp and skin of an individual and optionally maintained in contact for a number of hours and optionally rinsed. It is possible, for example, to apply the composition containing an effective amount of at least one compound of the invention in the evening, to retain the composition in contact overnight and optionally to shampoo in the morning. These applications can be repeated daily for one or a number of months, depending on the particular individuals involved.
Liposomes and emulsions are well known examples of delivery vehicles or carriers for hydrophobic drugs. Certain organic solvents such as dimethylsulfoxide also may be employed. Additionally, the compounds may be delivered using a sustained-release system, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent. Various sustained-release materials have been established and are well known by those skilled in the art. Sustained-release capsules may, depending on their chemical nature, release the compounds for a few weeks up to over 100 days. Depending on the chemical nature and the biological stability of the therapeutic reagent, additional strategies for stabilization may be employed.
Pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve their intended purpose, to effect a therapeutic benefit, or to effect a detectable change in the function of a cell, tissue, or organ. More specifically, a therapeutically effective amount means an amount effective to prevent the development of or to alleviate the existing symptoms of the subject being treated. Determining the effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.
Toxicity and therapeutic efficacy of the compounds or compositions can be determined by standard pharmaceutical, pharmacological, and toxicological procedures in cell cultures or experimental animals. For example, numerous methods for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population) exist. The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the ratio between LD50 and ED50. Compounds and compositions exhibiting high therapeutic indices are preferred. The data obtained from cell culture assays or animal studies can be used in formulating a range of dosages for use in humans. [See, for example, Fingl et al., in The Pharmacological Basis of Therapeutics, Ch. 1 p. 1 (1975)].
The compounds of the present invention may be administered by a single dose, multiple discrete doses or continuous infusion. Because the compounds preferably are non-peptidic, easily diffusible and relatively stable, they can be well-suited to continuous infusion.
Dose levels on the order of about 0.1 mg to about 10,000 mg of the active ingredient are useful in the treatment of the above conditions, with preferred levels being about 0.1 mg to about 1,000 mg. The specific dose level, and thus the therapeutically-effective amount, for any particular patient will vary depending upon a variety of factors, including the activity of the specific compound employed and its bioavailability at the site of drug action; the age, body weight, general health, sex and diet of the patient; the time of administration; the rate of excretion; drug combination; the severity of the particular disease being treated; and the form of administration. Typically, in vitro dosage-effect results provide useful guidance on the proper doses for patient administration. Studies in animal models also are helpful. The considerations for determining the proper dose levels are available to the skilled person.
Certain compounds can administered in lyophilized form. In this case, 1 to 1000 mg of a compound of the present invention may be lyophilized in individual vials, together with a carrier and a buffer, such as mannitol and sodium phospshate. The compound may be reconstituted in the vials with bacteriostatic water before administration.
In treating neurological disorders resulting from global or focal ischemia, for example, the compounds of the present invention are preferably administered orally, rectally, parenterally or topically at least 1 to 6 times daily, and may follow an initial bolus dose of higher concentration.
For the compounds, methods, and uses of the present invention, any administration regimen regulating the timing and sequence of drug delivery can be used and repeated as necessary to effect treatment. Such regimen may include pretreatment and/or co-administration with additional therapeutic agents.